“Guide” of muscone modification enhanced brain‐targeting efficacy and anti‐glioma effect of lactoferrin modified DTX liposomes

Abstract Glioma is one of the most aggressive malignant diseases for human health. It is difficult to resect completely due to their invasiveness. The targeted delivery, as a noninvasive approach, is a major strategy for the development of treatments for brain tumors. Lactoferrin (Lf) receptors are over‐expressed in both brain endothelial cells and glioma cells. Macromolecular Lf modified nanoparticles have been shown to enhance the brain targeting. Muscone is a “guide” drug that have been demonstrated to promote liposomes into the brain by modification. To further enhance the brain‐targeting efficacy of Lf modified carriers, we designed that Lf and muscone dual‐modified liposomes cross blood–brain barrier (BBB) and target to brain for enhanced docetaxel (DTX) brain delivery. The results showed that we successfully prepared Lf and muscone dual‐modified liposomes (Lf‐LP‐Mu‐DTX), the number of Lf molecules connected to the surface of per liposome was 28. Lf‐LP‐Mu‐DTX increased uptake in both U87‐MG cells and hCMEC/D3 cells, enhanced penetration of U87‐MG tumor spheroid and in vitro BBB model, had better in vitro and in vivo anti‐tumor effects. In conclusion, “guide” of muscone modification enhanced brain‐targeting efficacy of Lf modified liposomes, Lf and muscone dual‐modified docetaxel loaded liposomes present a potential brain‐targeting drug delivery system for use in the future treatment of gliomas.


| INTRODUCTION
Gliomas are the most common primary malignant tumors, accounting for about 80% in malignant brain tumors. 1 Since most gliomas grow invasively in the cerebral hemisphere and cannot be completely removed by the surgery, chemotherapy remains an important treatment for malignant glioma. However, the presence of blood-brain barrier (BBB) is the biggest obstacle to chemotherapy drugs. BBB strictly controls the delivery of plasma to solutes. 2 Almost all large-molecule and over 98% of small-molecule drugs cannot pass the BBB, and only lipid-soluble drugs with a molecular mass of less than 600 can cross the BBB via passive diffusion. 3 Recently, brain targeting delivery systems have represented a promising strategy to improve the treatment of glioma. Brain-targeted drug delivery systems deliver some therapeutic or diagnostic agent to the tumor site in brain, and enhance the accumulation of chemotherapeutic drugs in the tumor site. 4,5 Recently, natural proteins as drug carriers have advantages including nontoxicity, biodegradability, non-immunogenic, renewability, and targeting ability. 6 Protein-based carriers have been reported in the form of drug conjugates, drug addicts, protein-mediated/ modified nanoparticles, and tissue engineering scaffolds. 6,7 Lactoferrin (Lf) is a transferrin-binding cationic glycoprotein of the transferrin family with a molecular weight of 80 kDa. 8 Lf binds to a specific Lf receptor on the surface of BBB and glioma cells. 9 Compared to transferrin, Lf with low plasma concentrations can avoid interference with endogenous Lf. Some studies have shown that Lf-modified delivery systems can promote drugs across the BBB and enhance anti-glioma effect. The brain-targeted drug delivery system with Lf as ligand has significantly higher accumulation in the brain than that of Tf and OX26. 10 Su et al. have successfully developed Lf-modified NPs as a targeting vector to promote brain tissue uptake of doxorubicin and treat gliomas. 11 In addition, Song et al. have demonstrated that Lf-conjugated nanoparticles can deliver drug and imaging probes across endothelial cells to achieve the transport of drugs into brain. 12 Traditional Chinese Medicines have been practiced in the clinic for hundreds of years. Aromatic resuscitation drugs can guide drugs upward to the brain. Recently, borneol and musk have been developed for enhancing BBB penetration. 13,14 In addition, borneol could promote co-administration targeting nanoparticles to enhance the brain targeting efficiency. 15 Muscone, as the "guide" drug of Traditional Chinese Medicines aromatic resuscitation, with low molecular weight (MW, 238.42) and strong fat-solubility, can facilitate the permeability of some drugs cross the BBB into the brain. 16 Further investigation illustrates that the mechanism of muscone promoted the permeability of drugs cross the BBB by inhibiting the expression of P-glycoprotein (P-gp) and matrix metalloproteinase-9 (MMP-9) and relaxing the tight junctions between epithelial cells. 14 Recently, aromatic resuscitation drugs are used as ligands to prepare ligand-modified targeted drug delivery systems by bioconjugate techniques, and to enhance brain targeting effect. A study reported that borneol-modified PAMAM could increase doxorubicin concentration in the brain and enhance the treatment of glioma. 17 Another study reported that borneol or muscone were conjugated with bovine serum albumin (BSA) to prepare aromatic resuscitation drugs modified albumin-based nanoparticles, which can enhance drugs across the BBB for the treatment of glioma. 18 Our recent research found that muscone modification increased the brain targeting of antibody-modified liposomes in vivo. 4 To further enhance the brain-targeted delivery of macromolecules ligands Lf modified carriers, Lf and muscone dual-modified PEGylated liposome were applied as an active glioma-targeting drug delivery system. PEGylation prolongs its circulating half-life and Lf modification enhances its uptake into cancer cells via receptor-and adsorption-mediated transcytosis. 19 DTX is extracted from cedar paclitaxel needles and then semi-synthesized. DTX has the better antitumor effect, twice as effective as paclitaxel. DTX has also being reported for the treatment of breast, nonsmall cell lung, prostate cancer, and brain cancers. 5,20 In the present study, we aimed to develop Lf and muscone dualmodification loaded DTX brain-targeted delivery systems, to investigate their characterization, and to evaluate their uptake capacity and uptake mechanism, BBB penetration ability and tumor spheroid penetration ability, in vivo brain targeting and therapeutic efficacy of glio-

| Cells and animals
U87-MG glioma cells and immortalized hCMEC/D3 cells were purchased from Shanghai Guandao Biological Engineering Co., Ltd (Shanghai, China). All cells were cultured in DMEM medium with 10% FBS and 1% antibiotics (penicillin and streptomycin). All the cells were cultured at 37 C, with 5% CO 2 .
The male BALB/c nude mice (16-20 g) were obtained from the Shanghai Silaike Laboratory Animal Co, Ltd (Hunan China). All our experiments were performed in compliance with guidelines set by the Guilin Medical University Institutional Animal Care and Use Committee.  23 The phospholipid concentrations of liposomes were determined by the Stewart method. 24 The number of Lf bound to liposomes was tested by Bradford assay. 25

| Physicochemical characterization of liposomes
The average diameter and zeta potential of liposomes were measured by Zetasizer Nano ZS (ZEN3600, Malvern Instruments, UK), as described previously. 26 where C is the total dose of docetaxel (μg/ml), C 0 is the concentration of docetaxel encapsulated in liposomes (μg/ml), n is the dilution ratio, and M is the total weight of DTX-LP (mg).
The in vitro release DTX from liposomes was evaluated using the dialysis method as our described previously. 4

| In vitro hemolysis assay
Blood was taken from the terminal retro-orbital region of Wistar rats, and fibrinogen was removed using a capillary glass tube. Erythrocytes were washed five times with normal saline and were centrifuged at 1500 rpm for 10 min. Erythrocytes were resuspended in normal saline and were adjusted to 2% concentration (v/v), and 20 μl liposomes, 100 μl 2% erythrocytes dispersion, 100 μl normal saline (negative control), and 1% Triton X-100 (positive control) were mixed up to EPP S C H E M E 1 Schematic illustration of Lf-LP-Mu-DTX that crosses the BBB, target the glioma, and exert anti-glioma effects.
tube and incubated at 37 C in a shaking water bath for 3 h, centrifuged at 1500 rpm for 10 min and the supernatant was collected. The samples were determined at 550 nm wave-length using a spectrophotometer (UV-2802, Unico, USA). The hemolysis rate was calculated as reported. 28 Liposomes (20 μl), 100 μl 2% erythrocytes dispersion, 100 μl normal saline (negative control), and 1% Triton X-100 (positive control) were mixed up to EPP tube and incubated at 37 C in a shaking water bath for 30 min, take 20 μl samples and place it on glass slides, and the erythrocyte agglutination was observed under the microscope.

| Cytotoxicity assay
Cytotoxicity of the various liposomes on U87-MG cells was determined by the MTT assay. 29 In brief, U87-MG cells were seeded onto 96-well plates at a density of 1 Â Then the penetrating capacity of formulations in the tumor spheroids was observed under a confocal fluorescence microscope.

| Endothelial permeability test of hCMEC/D3 cells
The model of BBB constructed by hCMEC/D3 cells as reported by Lopalco et al. 35 Cells were cultured on a 12-transwell insert, transendothelial electrical resistance (TEER) and the quality of the cell monolayer was measured and compared to previously reported values. 36 Endothelial permeability of formulations across cell monolayers was carried out according to previously described protocols. 4 The apparent permeability coefficient (P app ) was calculated according to the following: where dM/dt is the cumulative measured fluorescence intensity in the receiver chamber per unit time, A is the surface area of the insert membrane, and C 0 is the initial concentration of coumarin 6 in the donor chamber. 37 The transport efficiency was calculated according to the following: The transport efficiency = the cumulative measured fluorescence intensity in the receiver chamber/initial fluorescence intensity in the donor chamber. 38 All experiments were performed in triplicate.

| In vivo imaging
The U87-MG in situ glioma models were established according to our previous reported. 4 On the 7th day after tumor implantation, three nude mice were randomly selected for magnetic resonance imaging (MRI) observation. The other brain glioma nude mice were randomly divided into four groups (n = 3), PEG-LP-DiR, Lf-LP-DiR, Lf-LP-Mu-DiR, Lf-LP-DiR + Mu (gavage), and the mice were injected 200 μl liposomes loaded DiR through the tail vein. After 24 h, the mice were killed and subjected to cardiac perfusion using normal saline and 4% paraformaldehyde, and then the brain, heart, liver, spleen, lung, and kidney were collected for further fluorescent imaging (Ex = 740 nm/Em = 790 nm).

| In vivo anti-glioma effect
We established an orthotopic U87-MG model in nude mice as above

| Statistical analysis
Eight mice were used per group for in vivo anti-glioma effect of animal study, and three mice were used per group for in vivo imaging of animal study. All experiments were performed in triplicate unless otherwise stated, and the results were presented as mean ± SD. Data were analyzed using the GraphPad Prism software. Differences between the two groups were evaluated by Student's t-test or oneway ANOVA analysis. Kaplan-Meier survival curves were used to present the differences in the survival of glioma-bearing mice with different treatments. Differences were considered statistically significant at p < 0.05 (*), and very significant at p < 0.01 (**).

| Preparation and characterization of LP
The liposomes with Lf and Muscone modification were prepared by thin-film dispersion method. The particle sizes and zeta potential of DTX-loaded liposomes were shown in Table 1   were consistent with about 5% hemolysis rates of traditional liposomes. 40 After different liposomes were incubated with erythrocytes, the morphology of erythrocytes in each group was shown in Figure 2. Compared with the normal saline group, PEG-LP, Lf-LP, and Lf-LP-M groups did not cause erythrocyte agglutination and erythrocyte abnormity. In general, the result showed that modified liposomes exhibited negligible toxicities on erythrocyte hemolysis assay and erythrocyte agglutination.

| Cytotoxicity assay
In vitro cytotoxicity of free DTX and different DTX-loaded liposomes on U87-MG cells was measured by MTT assay. As shown in When DTX was loaded into liposomes, which could be attributed to higher intracellular delivery of DTX from enhancing cell uptake, 41

| In vivo imaging
To confirm the in vivo targeting ability of liposomes, the in vivo distribution of DiR-loaded liposomes in the glioma-bearing nude mice was  Figure 7b, which had confirmed brain tumor formation.
After 24 h, the brain, heart, liver, spleen, lung, and kidney were collected for ex vivo imaging in Figure 7c. In Figure 7c, compared with the PEG-LP-DiR group, Lf-LP-DiR has been shown to enhance the delivery of the drug into the brain. The accumulation of the Lf-LP-Mu-DiR group in the brain tumor site was much higher than that in Lf-LP-DiR and Lf-LP-DiR + Mu groups. According to fluorescence imaging of the brain in Figure 7c, the fluorescence signal of the orthotopic glioma in different formulation groups was further semiquantitatively analyzed in Figure 7e, the region where we quantified the fluorescence signal of glioma was shown in Figure 7d. In Figure 7e, the fluorescence intensity of Lf-LP-DiR group is higher than that of PEG-LP-DiR group (p < 0.01), and the fluorescence intensity of Lf-LP-Mu-DiR group are higher than that of Lf-LP-DiR (p < 0.01) and Lf-LP-DiR + Mu group (p < 0.01), respectively. This is in agreement with the results of cellular uptake and tumor spheroid penetration.
For Lf-LP-DiR + Mu group, results show that the fluorescence intensity of Lf-LP-DiR + Mu group are significantly lower than that of Lf-LP-Mu-DiR group (p < 0.01) and somewhat higher than that of Lf-LP-DiR group. ing by adsorption-mediated endocytosis pathways. 19 Some studies reported that LfR-mediated endocytosis was unidirectional, which might increase the uptake of Lf-modified liposomes. 11,50 In the mechanism study of cellular uptake, from Figure 4f pathways. 32,42 And then, our results showed that free LF significantly reduced the cellular uptake of Lf-LP-Mu-C6 in U87 cells compared with control group (p < 0.01), which suggested the internalization of Lf-LP-Mu-C6 involved Lf receptor-mediated endocytosis, consistent with literature reported. 45 However, the results showed that muskone did not inhibit cellular uptake, but it can slightly increase cellular uptake. Therefore, we think that muskone does not have a specific blocking effect on endocytosis.

| In vivo anti-glioma effect
In the study of in vitro cytotoxicity, Lf-LP-Mu-DTX group showed the highest in vitro antitumor activity than other groups, which possibly was due to cellular uptake increasing of the formulations. The results were consistent with our previous report. 4 In ex vivo imaging from Figure  showed that the fluorescence intensity of Lf-LP-DiR + Mu group was significantly lower than that of Lf-LP-Mu-DiR group (p < 0.01) and somewhat higher than that of Lf-LP-DiR group. We speculated that muscone (gavage) had little effect on the lipophilicity of the targeted delivery system. However, muscone (gavage), as an aromatic resuscitation drug from Traditional Chinese Medicines, can promote Lf-LP-DiR to enter into the brain rapidly and increase Lf-LP-DiR elimination quickly, which may be due to the bidirectional permeation effect of muscone as previously reported. 4 The result of in vivo anti-glioma showed that the Lf-LP-Mu-DTX group exhibited the best tumor suppressive effect, which was in agreement with the results of ex vivo imaging. The result suggested that Lf and muscone dual modified liposome had more favorable brain targeting ability, enhanced in vivo anti-glioma effect, prolonged median survival time. In addition, the modified liposomes did not show systemic side effects.
In the process of the study, we found that some factors affected the efficacy of the targeted delivery system into the brain, including drug release, internalization, circulation, blood-brain barrier recognition, and so forth. 53 Changes in each process would affect the delivery efficiency of the brain-targeting drug delivery system. Only by ensuring that the targeted drug delivery system with minimum loss in each process, it can enter the brain more and exert its curative effect. For example, modified nanoparticles can absorb plasma protein by way of specific and nonspecific interactions in systemic administration, which alters their physiochemical properties and following interactions with targeted cells. 54 In traditional Chinese Medicines, aromatic resuscitation drugs were used to enhance the therapeutic effects of brain diseases and have a long history of application. In addition, the aromatic resuscitation drugs have good safety. 18 In the present study, we used muscone as the "guide" drug and Lf as the target ligand to design brain targeting delivery system, in order to achieve improving brain targeting and therapeutic effect of glioma by Lf and muscone dual modification. Our study results showed that LF and muscone dualmodified liposomes significantly enhanced brain targeting effect than by physical addition muscone group. Therefore, we think that muscone chemical modification is different from the physical addition of muscone on altering mechanisms of the BBB permeability.
The further mechanisms for muscone chemical modification need to be explored in future studies. Our study would provide a reference for the brain targeting application of aromatic resuscitation drugs in traditional Chinese Medicines.

| CONCLUSION
In conclusion, we established Lf and muscone dual-modified DTX long-circulating liposomes for glioma therapy, where Lf was modified for active targeting to LfR and muscone was modified for increasing the BBB permeability on the surface of the liposome. Lf and muscone dual-targeted liposomes significantly enhanced the cellular uptake of glioma cells, in vivo brain targeting effect, and in vitro and in vivo anti-